Characterization of Starch Polysaccharides
in Aqueous Systems:
de facto molar masses vs supermolecular structures
Werner Praznik and Anton Huber
Department of Chemistry
BOKU - University of Natural Resourses and Applied Life
Science Vienna / Austria
Institute of Chemistry
KF-University Graz / Austria
Content
• Introduction
• Analysis of molecular dimension by SEC-analysis by means of
multiple detection systems
• Synthetic amylose – ncb glucan
• Partially hydrolized starch scb glucan ( waxy maize, H 70 000)
• Potato starch glucans
• long-chain branched (lcb) - glucan / amylose
• short-chain branched (scb) - glucan / amylopectin
Kinds of starch glucans:
non-branched (nb) / long-chain branched (lcb)
glucans / amylose
symmetry / order: conformation of α -Helices
hydrophilic / hydrophobic domains
intra-molecular stabilizing: low
pronounced tendency for retrogradation,
gel formation (supramolecular structures)
(α(1→4)-D-Glc)30→ dp 30
instance / model : synthetic amylose
short-chain branched (scb) glucans /
amylopectin
(α(1→4)-D-Glc) with
α(1→6)linkages → dp 43
α(1→4) + α(1→6) linked branches
α(1→6) positions are symmetry breaker forming
irregular structures - more or less compact coils
no hydrophilic /-phobic domains
intra-molecular stabilizing: high
comparably increased packing density + solubility
instance / model:
partially hydrolized glucan from waxy maize
Molecular dimension by size exclusion chromatography (SEC)
Distribution of glucan excluded volume (Ve = ip . mdmc)
by means of size exclusion chromatography (SEC)
- an entropy controlled separation on gel columns systems
•
SEC – combined with specific molar detection
(e.g Fluorescence of an unique chromophor in a molecule)
and universal mass detection (SEC-mass/molar).
Information about molar mass of constituting glucans
•
SEC - combined with light scattering (LS)
and universal mass detection (SEC-mass/LS)
Sensitive towards high molecular components, in particular
towards glucan-aggregates
Information about supramolecular structure
Absolute Molar Mass Distribution
Quantitative labeling of terminal hemiacetals +
SEC - mass / molar detection: DRI / fluorescence λ380
700
600
500
400
300
200
100
0
32
34
36
38
V_ret [mL]
40
42
44
Absolute Molar Mass Distribution
SEC – mass / scattering intensity / excluded volume - detection
SEC - DRI / LS / viscosity
Synthetic amylose – ncb glucan
Synthesis of amylose by catalytic action of potato phosphorylase
H
OH
HO
CH2OH
O
H
H
H
H
H
OH
O
HO
H
CH2OH
HO
H
H
H
OH
H
O
HO
maltoheptaose
5
H
potato phosphorylase
OH
HO
citrate puffer
pH 6,1
CH2OH
O
H
H
H
OH
CH2OH
HO
H
1
OH
H
H
H
O
HO
OH
n
HO
H
O-
O
P
OH
H
O-
H
OH
CH2OH
HO
H
H
amylose
+
CH2OH
O
H
H
O
glu-1-P
OH
H
CH2OH
HO
H
1
OH
O
HO
H
(5 + n)
OH
+
H
n
-
O
P
O
OH
n = 1,2,3,...100,....
procedure:
1 g glucose-1-P and 4.5 mg maltoheptaose in citrat puffer pH 6.1 (50 mL)
+ 0.03 % NaN3 were incubated at 40° for 30 min.
Addition of 3 mL of chromatographically purificated potato phosphorylase and
incubation at 40°C.
For analysis equivalents were taken at increasing incubation times
O-
Synthesis of amylose by catalytic action of potato phosphorylase
D = M [ g / mol ] =
molar mass [Dalton]
starter of synthesis:
maltoheptaose 0,077 μmol/mL
70
60
% turnover
m[ g / L]
n[mol / L]
determination of free phosphat:
50
40
calculation of turnover of glucose –1 – P
30
calculation of mass of amylose μg / mL
20
3 h synthesis: 59% turnover - 5590 μg amylose / mL
10
M = 5590 μg /0,077μmol
0
0
1
2
3
4
5
time h
= 72 600 [g/mol]
DRI
200
M = 3450 μg /0,077μmol
= 44 805 [g/mol]
160
[mV]
1 h synthesis: 36% turnover - 3450 μg amylose / mL
120
Glu-1-P
synthetic amylose
citrate puffer
80
40
0
20
30
40
50
V_ret [mL]
60
70
Synthesis of amylose by catalytic action of potato phosphorylase
SEC –analysis of synthetic amyloses:
3.5
160
[mV]
120
80
Glu-1-P
synthetic amyloses
citrate puffer
40
massfraction
fraction
mass
mass/molar
fraction
DRI
2.8
2.1
1.4
0.7
0
0
20
30
40
50
V_ret [mL]
period of synthesis
h
% turnover
60
70
0
30000
60000
90000
120000
M [g/M]
SEC-results
turnover calculated results
Mw
Mn
Mw/ Mn
Mn
1
36
54 000
50 000
1.1
44 800
3
59
84 000
76 000
1.1
72 600
4
59
82 000
74 000
1.1
72 800
150000
Molecular dimension of synthetic amyloses
• Quantitative pyridylamination of terminal hemi acetal groups of
amylose = AP-amyloses
• SEC-analysis of AP-amylose by means of mass (RI) and molar
(flourescence) detection: dissolved in DMSO/water.
SEC- eluent : 0.05 M NaCl
• SEC-profil analysis of AP-amyloses by mass (RI), scattering intensity
(LS) and viscosity detection: dissolved in DMSO/water/ 0.005 M Na2CO3.
SEC-eluent: 0.005 M Na2CO3
Quantitative pyridylamination of terminal hemi-acetal group of amylose
O
O
CH2OH
O
amylose
H
O HO
OH
n
OH
+
H2 N
N
aminopyridine
CH2OH
O
- H2O
H
O
O
amylose-imid
OH
H
O HO
H
H
H
n
C
OH
N
N
CH2OH
O
+ H2
H
O
H
O
Na-cyanoborhydride
AP-amylose
OH
H
O HO
H
n
OH
H2
C
NH
N
Procedure: Part of fresh centrifugated amyloses (precipitate from solution of synthesis +
10% of n-BuOH) + 1 mL H2O + 1g aminopyridine + 0.7mL conc. HCl (pH=7) stirred at 62°C
for 24 h. Then 0.2g NaBH3CN was added and further stirred at 62°C for 24 h.
Part of the solution added to MeOH and precipitated AP-glucan were purified with MeOH
and acetone. Precipitates were redissolved in 2 mL DMSO + 0.5 mL H2O for SEC-analysis.
SEC-analysis of AP-amylose by means of universal mass (DRI)
and molar concentration (flourescence 380 nm) detection
3.5
ev_mass
mass/molar fraction
DRI-detection
0.25
0.2
0.15
FD - 380 nm
0.1
0.05
2.8
2.1
1.4
0.7
0
21
28
35
V_ret [mL]
42
49
30000
60000
90000
120000
150000
M [g/M]
5
4.9
4.8
0.18
4.7
0.12
4.6
0.06
4.5
0
4.4
20
25
30
V_ret
35
[mL]
40
45
lg(M)
mass fraction
0.24
10 mg of AP amylose solved in 2 mL DMSO
24h at 62°C+ 0.5mL eluent , 300µl Injection
RI 8x, FD – 380nm att.10
column system: Superose12+Superose 6 +
Fractogel HW 40 eluent : 0.05 M NaCl
Mw= 87 000 g/mol
Mn = 54 000 g/mol
Mw/Mn = 1.6
mass fraction
1.2
0.9
0.6
0.3
4
5
6
7
8
V_ret [ml]
9
10
11
Column system:
Superdex 75 (10x300mm);
eluent: =0.005
Na2CO3/0.05M NaCl;
120
90
60
30
6.4
7.2
8
8.8
9.6
V_ret [ml]
scattering intensity 5° [g/ (mL mol )]
intrinsic viscosity [mL/g]
SEC-profil analysis of AP-amylose by means of mass (DRI),
viscosity and scattering intensity (LALLS) detection
800000
600000
400000
200000
4
5
6
7
8
9
V_ret [ml]
Mean Intrinsic Viscosity
[η] = 70 mL/g
corresponds roughly with
constituting molecules
bulk analysis of AP-amylose: scattering intensity at low
angle of detection (5°) – sensitive information about
glucan-aggregation / supramolecular structures
apparent Mw = 500 000 g/mol – 10 times the constituting molecules
supramolecular structures in the applied aqueous system
10 11
Partially hydrolized starch scb glucan
( waxy maize, H 70 000 )
Partially hydrolized starch glucan ( waxy maize, H 70 000 )
short chain branched glucan (scb glucan)
50 mg scb glucan were solved in 5 mL 0.05M NaCl + 0.3 mL n-Butanol stirring @ 65°C ,
40h. Then + 0.2mL n-Butanol were added , stirring @ room temperature, 5 h and cool
storage @ 5-6°C, 5h. Not any precipitation
0.24
5.5
0.18
5
0.12
4.5
Column system: Superose 12 (10x300mm)
+ Superose 6 (10x300mm) + Fractogel
HW40 (10x300mm); eluent: 0.05M NaCl;
DRI- and Fl (315nm/380nm)-detection
lg(M)
mass fraction
scb-glucan was precepitated with MeOH:Aceton (1:1), washed with MeOH/Aceton
and derivatisied with aminopyridine. AP-amylopectin sample for SEC was solved in 1mL
DMSO + 0.5 mL water.
Mw= 121 000
0.06
4
Mn = 50 400
Mw/Mn = 2.4
0
20
25
30
V_ret
35
40
45
[mL]
AP scb glucan solved in 1 mL DMSO +0.5mLH2O @
65°C 8h. Profil of normalized mass fractions, area = 1.0;
calibration with absolut mass/molar detection
Partially hydrolized starch glucan ( waxy maize, H 70 000)
short chain branched glucan (scb glucan)
mass (DRI) and LALLS detection: solved in DMSO/water/ 0.005 M Na2CO3
SEC-mass/molar detection
1.4
Mw= 94 000 g/mol
Mn = 50 400 g/mol
Mw/Mn = 1.9
mass fraction
1.2
1
SEC-mass/LALLS detection
0.8
Mw=152000 g/mol
Mn=53700 g/Mol
Mw/Mn=2.8
0.6
0.4
0.2
0
3.5
4
4.5
lg(M)
5
[g/mol]
5.5
6
Mean Intrinsic Viscosity
[η] = 30 mL/g
corresponds roughly to
constituting molecules
Mw similar for both approaches
minor supramolecular structures
Potato starch glucans
lcb glucan (amylose)
scb glucan (amylopectin)
SEC-analysis of potato starch glucans: lcb glucan (amylose) + scb
glucan (amylopectin) precipitate with MeOH
Mw =741 000 Mn = 81 500
Mw/Mn = 9.1
100 µm
Potato starch disssolved
in DMSO/eluent/nButanol,
precipitated with MeOH
and derivatizied with
aminopyridine.
mass fraction
0.2
6
5.6
5.2
0.15
4.8
lg(M)
0.25
0.1
4.4
0.05
4
0
3.6
20
25
30
V_ret
35
40
45
[mL]
AP- glucans dissolved in 0.3 mL DMSO +0.7mLeluent @ 65°C 20h. Profil of normalized
mass fractions, area = 1.0; calibration with absolut mass/molar detection
Column system: Superose 12 (10x300mm) + Superose 6 (10x300mm) + Fractogel HW40
(10x300mm); eluent: 0.05M NaCl
Iodine staining of potato starch fractions
native potato starch
Preparation of potato starch glucans after aqueous dissolving
50 mg potato starch were dissolved in 5 mL 0.05M
NaCl + 0.3 mL n-Butanol stirring @ 65°C , 40h.
Potato starch
100 µm
Then + 0.2mL n-Butanol were added , stirring @ room
temperature, 5 h and cool storage @ 5-6°C, 5h.
Precipitate of lcb glucan (amylose) - centrifuged @
13000 rpm, 10 min. Precipitate washed three times
with n-Bu/water and derivatizied with aminopyridine.
AP-amylose sample for SEC was solved in 1mL DMSO
+ 0.5 mL water.
Amylopectin in supernatant – precepitated with
MeOH Precipitate washed three times with MeOH and
derivatizied with aminopyridine. AP-amylopectin sample
for SEC was dissolved in 1mL DMSO + 0.5 mL water.
SEC-analysis of potato starch glucans: lcb glucan (amylose)
precipitate with n-Butanol
0.24
5.5
0.18
5
0.12
4.5
0.06
4
lg(M)
mass fraction
Mw =123 000 Mn = 56 000
Mw/Mn = 2.2
0
20
25
30
V_ret
35
40
45
[mL]
AP- lcb glucan (amylose) solved in 1 mL DMSO +0.5mLH2O @ 65°C 8h.
Profil of normalized mass fractions, area=1.0; calibration with absolut mass/molar
detection; Column system: Superose 12 (10x300mm) + Superose 6 (10x300mm) +
Fractogel HW40 (10x300mm);
Iodine staining of potato starch fractions
lcb glucan (amylose)
precipitate with n-Butanol
SEC-profil analysis of AP- lcb glucan (amylose) with mass
(DRI) and LALLS detection: solved in DMSO/water/ 0.005 M
Na2CO3
bulk analysis of potato AP-amylose with low
angle scattering
information about glucan-aggregates building
process - supramolecular structure
apparent Mw = 3 000 000 g/mol
30 times the constituting molecules
supramolecular structures in the
applied aqueous system
Column system: Superdex 75
(10x300mm); eluent: =0.005
Na2CO3/0.05M NaCl;
Mean Intrinsic viscosity [η] = 80 mL/g
roughly corresponds with constituting
molecules
SEC-analysis of potato starch glucans: scb glucan (amylopectin)
precipitate with MeOH
Mw =242 000 Mn = 210 000
Mw/Mn = 1.2 78%
0.24
5.7
0.18
Mw =70 000 Mn = 36 000
Mw/Mn = 1.9 22%
5.1
0.12
4.8
0.06
4.5
lg(M)
mass fraction
5.4
4.2
0
20
25
30
V_ret
35
40
45
[mL]
AP- scb glucan (amylopectin) solved in 1 mL DMSO +0.5mLH2O @ 65°C 8h.
Profil of normalized mass fractions, area=1.0; calibration with absolut mass/molar
detection; Column system: Superose 12 (10x300mm) + Superose 6 (10x300mm) +
Fractogel HW40 (10x300mm);
Iodine staining of potato starch fractions
scb glucan (amylopectin)
precipitate with MeOH
SEC-profil analysis of AP- scb glucan (amylopectin) with
mass (DRI) and LALLS detection: solved in DMSO/water/
0.005 M Na2CO3
bulk analysis of potato AP-amylopectin with low
angle scattering
information about glucan-aggregates building
process - supramolecular structure
apparent Mw = 57 000 000 g/mol
300 times the constituting molecules
supramolecular structures in the
applied aqueous system
Column system:
Superdex 75 (10x300mm);
eluent: =0.005 Na2CO3/0.05M NaCl;
Mean Intrinsic viscosity [η] = 70 mL/g
Roughly corresponds to constituting
molecules
Conclusion:
ƒ Instance / Model for ncb-glucan (synthetic amylose) forms
supramolecular structures (aggregates) in aqueous systems more
or less immediately → high tendency for inter-molecular interaction.
ƒ Instance / Model for low molar mass scb-glucan (partially
hydrolized waxy maize starch) forms minor supramolecular
structures in aqueous systems → preferred intra-molecular
interaction and intra-molecular stabilization.
ƒ de facto molar mass of potato lcb-glucan (native amylose) from
molar mass distribution: Mw = 123 000, Mn = 56 000, Mw/Mn = 2.2;
tendency to form supramolecular structures is very high; apparent
Mw from light scattering → 3 000 000 g/Mol.
ƒ Potato scb-glucan (native amylopectin) is partially dissolved in
applied solvent system only; molar mass in the range 20 000 - 300
000 g/Mol; a significant fraction (approx. 70%) of molecules gets not
dissolved truely in the aqueous system and remain / form
supermolecular structures: apparent Mw LS → 57 000 000 g/Mol.